Figure 6.
Figure 6. TF-FVIIa signaling in A7 melanoma cells requires FVIIa-mediated integrin complex formation and arf6 function. (A) TF-integrin β1 interaction in TF and PAR2 transduced A7 melanoma cells that were stimulated for indicated times with FVIIa (10 nM). Left panel: representative western blots of TF and integrin β1 in immunoprecipitates of Brij 35 cell lysates using DynaBead-coupled TS2/16 or AIIB2 antibody. Right panel: densitometric quantification of western blots (n = 4). *P < .05, **P < .01, ***P < .001, one-way ANOVA and Dunnett’s test. (B) Representative western blots and densitometric quantification of 4 independent experiments of TF in a TS2/16 pull-down assay from Brij 35 lysates from A7 cells stimulated with 10 nM FVIIa wt or E26A (n = 4). **P < .01, ***P < .001. One-way ANOVA and Tukey’s test. (C) Quantification of IL-8 mRNA in A7 cells after 90 minutes of stimulation with FVIIa wt (10 nM) or E26A (10 nM) (n = 7). *P < .05, **P < .01, ***P < .001, ANOVA multiple comparison with Bonferroni correction. (D) Western blots of TF, integrin β1, arf6, and HA-tag of A7 cell lysates transduced to express HA-tagged arf6 wt, constitutively active mutant Q67L, or dominant-negative mutant T27N. (E) Cell surface expression of TF detected by antibody 5G9 in TF and PAR2 transduced cells co-expressing arf6 wt, Q67L, or T27N. Expression levels were normalized to TF and PAR2 transduced cells without arf6 virus from the same experiment (n = 5). *P < .05, one-way ANOVA and Tukey’s test. (F) FXa generation on A7 cells expressing arf6 mutants (n = 3). *P < .05, ** P < .01, one-way ANOVA and Dunnett’s test. (G) IL-8 mRNA quantification in A7 cells expressing arf6 mutants after 90 minutes of stimulation with FVIIa (10 nM) or SLIGRL (50 μM) (n = 4). **P < .01, one-way ANOVA and Tukey’s test. (H) Trans-well migration assay of highly motile M24met melanoma cells that were left untreated (black bars) or pre-incubated with SH3 (20 μM; white bars) before stimulation with 10 or 50 nM FVIIa (n = 3). *P < .05, Student t test. (I) Trans-well migration assay of M24met melanoma cells stimulated with 10 nM or 50 nM FVIIa wt and E26A (n = 3). **P < .05, one-way ANOVA and Tukey’s test.

TF-FVIIa signaling in A7 melanoma cells requires FVIIa-mediated integrin complex formation and arf6 function. (A) TF-integrin β1 interaction in TF and PAR2 transduced A7 melanoma cells that were stimulated for indicated times with FVIIa (10 nM). Left panel: representative western blots of TF and integrin β1 in immunoprecipitates of Brij 35 cell lysates using DynaBead-coupled TS2/16 or AIIB2 antibody. Right panel: densitometric quantification of western blots (n = 4). *P < .05, **P < .01, ***P < .001, one-way ANOVA and Dunnett’s test. (B) Representative western blots and densitometric quantification of 4 independent experiments of TF in a TS2/16 pull-down assay from Brij 35 lysates from A7 cells stimulated with 10 nM FVIIa wt or E26A (n = 4). **P < .01, ***P < .001. One-way ANOVA and Tukey’s test. (C) Quantification of IL-8 mRNA in A7 cells after 90 minutes of stimulation with FVIIa wt (10 nM) or E26A (10 nM) (n = 7). *P < .05, **P < .01, ***P < .001, ANOVA multiple comparison with Bonferroni correction. (D) Western blots of TF, integrin β1, arf6, and HA-tag of A7 cell lysates transduced to express HA-tagged arf6 wt, constitutively active mutant Q67L, or dominant-negative mutant T27N. (E) Cell surface expression of TF detected by antibody 5G9 in TF and PAR2 transduced cells co-expressing arf6 wt, Q67L, or T27N. Expression levels were normalized to TF and PAR2 transduced cells without arf6 virus from the same experiment (n = 5). *P < .05, one-way ANOVA and Tukey’s test. (F) FXa generation on A7 cells expressing arf6 mutants (n = 3). *P < .05, ** P < .01, one-way ANOVA and Dunnett’s test. (G) IL-8 mRNA quantification in A7 cells expressing arf6 mutants after 90 minutes of stimulation with FVIIa (10 nM) or SLIGRL (50 μM) (n = 4). **P < .01, one-way ANOVA and Tukey’s test. (H) Trans-well migration assay of highly motile M24met melanoma cells that were left untreated (black bars) or pre-incubated with SH3 (20 μM; white bars) before stimulation with 10 or 50 nM FVIIa (n = 3). *P < .05, Student t test. (I) Trans-well migration assay of M24met melanoma cells stimulated with 10 nM or 50 nM FVIIa wt and E26A (n = 3). **P < .05, one-way ANOVA and Tukey’s test.

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